Beta cell transplantation holds great promise to improve treatment of Type 1 diabetes but a number of obstacles need to be overcome first. Among these is the scarcity of available donor islets. Embryonic stem (ES) cell derived beta cells can in principle supply unlimited numbers of beta cells for transplantation but reliable protocols for generating fully functional beta cells are not yet developed. Formation of definitive endoderm (DE) cells from embryonic stem cells has been reported for both mouse and human ES cells in, e.g., WO 2005/116073, WO 2005/063971, and US 2006/0148081. Efficient generation of pancreatic endoderm (PE) cells from, e.g., DE cells is advantageous for generation of insulin-producing beta cells for the treatment of diabetes.
In attempting to cultivate fully differentiated pancreatic Islet cells, the objective has long been to isolate pancreatic cells including pancreatic endocrine pre-progenitor cells that are capable of differentiating into pancreatic beta cells or islets. One important step in isolation of the pancreatic endocrine lineage from the exocrine lineage would be to identify recognizable cell markers, specific for the pancreatic endocrine pre-progenitor cells and/or progeny thereof. Both intracellular and extracellular markers have been investigated for this purpose. Intracellular markers, particularly transcription factors detected in embryonic pancreatic cells that develop into fully differentiated Islet cells, have been extensively studied as progenitor markers. In some aspects these intracellular markers are transcription factors detected in embryonic pancreatic cells that develop into mature islet cells. Transcription factors such as Pdx1, Ngn3, Pax6, and Isl1, for example, have been studied. They are expressed in cells that are programmed during embryonic development to become pancreatic endocrine cells. However, these intracellular markers offer less practical value than extracellular markers, because analysis of expression of those markers requires either the killing of the cells or permanent modification of the cells by genetic engineering of reporter genes into the cells.
In particular, the earliest multipotent stem or progenitor cells comprising the early pancreatic bud structures (found at day 9.5-10.5 of mouse fetal development) co-express 3 transcription factors: Pdx1, Nkx6.1 and Ptf1a. The expression of Ptf1a and Nkx6.1 then segregate into the peripheral acinar committed domain and the central ductal/endocrine committed domain, respectively. See Hald et al., J Histochem Cytochem (2008); 56(6):587-95. It is thus of high priority to identify useful surface markers that are selectively expressed in pancreatic tissue subsets, such as the particular subpopulation of early “ductal/endocrine” progenitors that already committed to chose the endocrine lineage. We have used the nomenclature for these cells as “endocrine pre-progenitors”. At this stage DNER is selectively expressed on the surface of endocrine pre-progenitor cells. Such cells will later express Ngn3 and progress towards endocrine maturation.
Once identified, extracellular markers would offer the advantage that the cells expressing the marker can be sorted under sterile conditions and kept alive. Epithelial cell adhesion molecules such as Ep-CAM and integrins have been investigated as pancreatic Islet progenitor markers. See, e.g., Cirulli et al., J. Cell Biol. 140: 1519-1534 (1998); and Cirulli et al., J. Cell Biol. 150: 1445-1460 (2000).